Abstract
Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR.
Highlights
It is well established that ligand binding and cell surface crosslinking of transmembrane proteins can lead to the assembly of large multicomponent protein complexes [1,2,3]
Related to its intracellular functions, Gal-3 has been identified as a component of heterogeneous nuclear ribonuclear protein [7], a factor in pre-mRNA splicing [8], and has been found to control cell cycle and prevent T cell apoptosis [9], whereas extracellular Gal-3 has been demonstrated to function in activating various types of inflammatory cells or mediating cell-cell and cell-extracellular matrix interactions [2,10,11]
Gene ontology (GO) annotations assigned thirteen (52%) to a nuclear or cytoplasmic location, five (20%) to the cytoskeleton or structural components, two (8%) proteins were predominantly secreted (a2 macroglobulin, galectin-3 binding protein), and five (20%) proteins were attributed to the cell membrane (CD147, integrin a3, integrin b1, monocarboxylate transporter-4, galectin-3)
Summary
It is well established that ligand binding and cell surface crosslinking of transmembrane proteins can lead to the assembly of large multicomponent protein complexes [1,2,3] While in this respect protein-protein interactions have been well studied in the recent years, there is an increasing awareness that ligand binding to information stored in cell surface glycans can lead to the assembly of large component protein complexes and modulate transmembrane signaling [4,5]. Galectins belong to the large family of lectins which bind to oligosaccharide complexes via beta (b)galactoside moieties. Among these the 30 kDa member Galectin-3 (Gal-3) is unique in that it is composed of a C-terminal carbohydrate recognition domain and an N-terminal non-carbohydrate-binding domain that facilitates its multimerization [6]. Related to its intracellular functions, Gal-3 has been identified as a component of heterogeneous nuclear ribonuclear protein (hnRNP) [7], a factor in pre-mRNA splicing [8], and has been found to control cell cycle and prevent T cell apoptosis [9], whereas extracellular Gal-3 has been demonstrated to function in activating various types of inflammatory cells or mediating cell-cell and cell-extracellular matrix interactions [2,10,11]
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